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Complete genome sequence of Stackebrandtia nassauensis type strain (LLR-40K-21T)
Standards in Genomic Sciences volume 1, pages 292–299 (2009)
Stackebrandtia nassauensis Labeda and Kroppenstedt (2005) is the type species of the genus Stackebrandtia, and a member of the actinobacterial family Glycomycetaceae. Stackebrandtia currently contains two species, which are differentiated from Glycomyces spp. by cellular fatty acid and menaquinone composition. Strain LLR-40K-21T is Gram-positive, aerobic, and nonmotile, with a branched substrate mycelium and on some media an aerial mycelium. The strain was originally isolated from a soil sample collected from a road side in Nassau, Bahamas. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of the actinobacterial suborder Glycomycineae. The 6,841,557 bp long single replicon genome with its 6487 protein-coding and 53 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
Strain LLR-40K-21T (=DSM 44728 = NRRL B-16338 = JCM 14905) is the type strain of Stackebrandtia nassauensis, which is the type species of the genus Stackebrandtia . S. nassauensis was originally isolated by M. P. Lechevalier and subsequently described by Labeda and Kroppenstedt  during the course of a 16S rRNA survey of putative Glycomyces strains. The genus was named after Erko Stackebrandt, a German microbiologist of note, who has contributed significantly to the molecular systematics the Actinobacteria. At present the genus Stackebrandtia contains only one additional species: S. albiflava, isolated from a soil sample collected from a tropical rainforest in China . Here we present a summary classification and a set of features for S. nassauensis strain LLR-40K-21T together with the description of the complete genomic sequencing and annotation.
Classification and features
A search of GenBank revealed no 16S rRNA reference sequences that were closely related to S. nassauensis. With 95% sequence similarity, the type strain S. albiflava, YIM 45751 , is the only cultivated strain with a sequence similarity above 91%, whereas a 16S rRNA gene sequence derived from a sample isolated from pig slurry (pig saw dust spent bedding in France, M982657, Snell-Castro et al., unpublished), represents the only related phylotype (with the same degree of sequence similarity as YIM 45751). Curiously, the type strains of the neighboring genus Glycomyces  were not within the 250 top hits in BLAST searches, with the 16S rRNA of type species G. harbinensis  sharing only 89% sequence similarity. Screening of environmental genomic samples and surveys reported at the NCBI BLAST server also showed no closely related phylotypes (with 93% sequence identity at the maximum), indicating a rather limited environmental occurrence of the species S. nassauensis (as of July 2009).
Figure 1 shows the phylogenetic neighborhood of S. nassauensis in a 16S rRNA based tree. The two 16S rRNA gene sequences in the genome of strain LLR-40K-21T are identical and do not differ from the previously published 16S rRNA sequence generated from NRRL B-16338 (AY650268).
S. nassauensis strain LLR-40K-21T cells are non-motile and filamentous, producing a with pale yellow to pale tan substrate mycelium on solid media  (Table 1 and Figure 2). Aerial mycelia are produced on some media and are white to yellowish-white in color . Both aerial and substrate mycelia are approximately 0.5 µm in diameter . Fragmentation of aerial or substrate mycelia into chlamydospores or zoospores has not been observed . Cells stain Gram-positive, grow aerobically, and are non acid-fast . Growth occurs at the temperature range of 15–37° C and in the presence of 4–9% NaCl. S. nassauensis LLR-40K-21T is positive for hydrolysis or degradation of allantoin, casein, esculin, gelatin, hypoxanthine, starch and tyrosine but negative for adenine and xanthine . The strain is capable of producing phosphatase and reducing nitrates; assimilation of acetate and malate is possible but not of benzoate, citrate, lactate, mucate, oxalate, propionate, succinate and tartarate . Acid is produced aerobically from arabinose, cellobiose, dextrin, fructose, galactose, glucose, glycerol, lactose, maltose, mannose, melibiose, methyl α-D-glucoside, raffinose, rhamnose, salicin, sorbitol, sucrose, trehalose and xylose; but not from adonitol, dulcitol, erythritol, inositol, mannitol, melezitose or methyl-β-xyloside .
The murein of S. nassauensis strain LLR-40K-21T contains meso-diaminopimelic acid as the diamino acid and N-glycolylmuramic acid as is found in members of the genus Glycomyces. Ribose is the major cell wall sugar. Mannose has also been reported [1,2]. Reports about the presence of inositol, arabinose, xylose and glucose differ [1,2]. Galactose, which has been identified in all Glycomyces strains, has not been found in Stackebrandtia . The fatty acid pattern of LLR-40K-21T is dominated by saturated branched chain acids, anteiso-(ai-) C17:0 (26.8%), ai-C15:0 (2.8%), and iso-(i-) C15:0 (8.7%), i-C16:1 (2.1%), i-C16:0 (8.7%), i-C17:0 (9.0%). Unsaturated straight chain acids play only a limited role: C17:1 cis9 (1.8%), and C16:1 cis9 (3.1%). A significant amount of ai-C17:0 2-OH (14.5%) and moderate amounts of hydroxylated fatty acids were also detected. Moderate amounts of saturated components including 10-methyl-branched heptadecanoic acid C16:010 methyl (9.0%) and 10-methyl-C17:0 (1.4%) were also detected. The occurrence of 10-methyl branched heptadecanoic acid and i-branched 1-OH fatty acids is differential for S. nassauensis from members of the genus Glycomyces which lack these acids. Polar lipids identified are phosphatidylglycerol, diphosphatidylglycerol, like in members of the genus Glycomyces, and two additional yet unknown phospholipids are present. Phosphatidylinisitolmanosides (PIM) and phosphatidylglycerol (PI), which are present in the members of the genus Glycomyces, are absent; however, PI is present in S. albiflava . Phosphatidylethanolamine (PE) and phosphatidylmethyl-ethanolamine (PME) were initially reported as absent in strain LLR-40K-21T , but were later observed by Wang et al. . The predominant menaquinones are MK-10 (H4), MK-10 (H6), MK-11 (H4) and MK-11 (H6), different from the patterns observed from the members of the genus Glycomyces which contain menaquinones with 9–12 isoprene units with various degrees of hydrogenation . Mycolic acids are absent .
Genome sequencing and annotation
Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position, and is part of the Genomic Encyclopedia of Bacteria and Archaea project. The genome project is deposited in the Genomes OnLine Database  and the complete genome sequence in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.
Growth conditions and DNA isolation
S. nassauensis strain LLR-40K-21T, DSM 44728, was grown in DSMZ medium 553 (GPHF Medium)  at 28°C. DNA was isolated from 1–1.5 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) with lysis modification DALM according to Wu et al. .
Genome sequencing and assembly
The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website. 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 7,157 overlapping fragments of 1,000 bp and entered into assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones . Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 308 Sanger finishing reads were produced to close gaps and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. The final assembly consists of 81,931 Sanger and 851,638 pyrosequence reads. Together all sequence types provided 40.0× coverage of the genome.
Genes were identified using Prodigal  as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline . The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform .
The genome is 6,841,557 bp long and comprises one circular chromosome with a 68.1% GC content (Table 3 and Figure 3). Of the 6,450 genes predicted, 6,487 were protein coding genes, and 53 RNAs; One hundred eight pseudogenes were also identified. The majority of the protein-coding genes (66.8%) were assigned a putative function while those remaining were annotated as hypothetical proteins. The properties and the statistics of the genome are summarized in Table 3. The distribution of genes into COGs functional categories is presented in Table 4.
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We would like to gratefully acknowledge the help of Susanne Schneider (DSMZ) for DNA extraction and quality analysis. This work was performed under the auspices of the US Department of Energy’s Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396. German Research Foundation (DFG) INST 599/1-1 supported DSMZ, and the Indian Council of Scientific and Industrial Research provided a Raman Research Fellow to Shanmugam Mayilraj.
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Munk, C., Lapidus, A., Copeland, A. et al. Complete genome sequence of Stackebrandtia nassauensis type strain (LLR-40K-21T). Stand in Genomic Sci 1, 292–299 (2009). https://doi.org/10.4056/sigs.47643
- mycelium producing
- 2-hydroxy fatty acids-containing